www.nature.com/scientificreports OPEN Taxonomic and functional characterization of a microbial community from a volcanic Received: 22 February 2019 Accepted: 24 July 2019 englacial ecosystem in Deception Published: xx xx xxxx Island, Antarctica Emma Martinez-Alonso1, Sonia Pena-Perez2, Sandra Serrano2, Eva Garcia-Lopez2, Alberto Alcazar1 & Cristina Cid2 Glaciers are populated by a large number of microorganisms including bacteria, archaea and microeukaryotes. Several factors such as solar radiation, nutrient availability and water content greatly determine the diversity and abundance of these microbial populations, the type of metabolism and the biogeochemical cycles. Three ecosystems can be diferentiated in glaciers: supraglacial, subglacial and englacial ecosystems. Firstly, the supraglacial ecosystem, sunlit and oxygenated, is predominantly populated by photoautotrophic microorganisms. Secondly, the subglacial ecosystem contains a majority of chemoautotrophs that are fed on the mineral salts of the rocks and basal soil. Lastly, the englacial ecosystem is the least studied and the one that contains the smallest number of microorganisms. However, these unknown englacial microorganisms establish a food web and appear to have an active metabolism. In order to study their metabolic potentials, samples of englacial ice were taken from an Antarctic glacier. Microorganisms were analyzed by a polyphasic approach that combines a set of -omic techniques: 16S rRNA sequencing, culturomics and metaproteomics. This combination provides key information about diversity and functions of microbial populations, especially in rare habitats. Several whole essential proteins and enzymes related to metabolism and energy production, recombination and translation were found that demonstrate the existence of cellular activity at subzero temperatures. In this way it is shown that the englacial microorganisms are not quiescent, but that they maintain an active metabolism and play an important role in the glacial microbial community. Ice from glaciers occupies approximately 11% of the Earth’s surface1. Although glaciers were traditionally con- sidered to be an uninhabitable environment, it has been proven that they are populated by a large number of microorganisms including bacteria, archaea and microeukaryotes2,3. Among them, microorganisms inhabiting the Antarctic glaciers are much more unknown than those living in other frozen environments4. Tree ecosys- tems can be diferentiated in glaciers: supraglacial, subglacial and englacial ecosystems5–7 (Fig. 1). Each ecosystem experiences diferent environmental conditions including temperature, solar radiation and nutrient content8,9. Microorganisms from the supraglacial and subglacial ecosystems have been the most widely studied. Among the three layers of a glacier, the upper layer (supraglacial ecosystem) is the most habitable, due to the atmospheric deposition of dissolved organic carbon, besides the plant and animal remains, and the particles and black carbon from atmospheric dust10. Te supraglacial ecosystem is largely populated by autotrophic micro- organisms exploiting the ample solar radiation and liquid water, and abundant nutrients available during ice-melt episodes11–13. Te subglacial ecosystem is dominated by aerobic chemoheterotrophs and by anaerobic methano- gens, nitrate reducers and sulfate reducers14–16 in basal bedrock and subglacial lakes. As there is no sunlight, these microorganisms obtain energy from the inorganic compounds originated from weathering and erosion of subgla- cial rocks17,18. Little is known about the persistence, physiology, and ecology of microbial communities inhabiting 1Department of Investigation, Hospital Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria, 28034, Madrid, Spain. 2Microbial Evolution Laboratory, Centro de Astrobiologia (CSIC-INTA), 28850 Torrejón de Ardoz, Madrid, Spain. Correspondence and requests for materials should be addressed to C.C. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:12158 | https://doi.org/10.1038/s41598-019-47994-9 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Ecosystems of a glacier colonized by microorganisms. Te supraglacial ecosystem is inhabited by photoautotrophic microorganisms, and by heterotrophic bacteria and microeukaryotes that feed on organic particles from atmospheric deposition. Microorganisms in the englacial ecosystem can be chemoautotrophs or heterotrophes that feed of solubilized products. Subglacial ecosystem contains a majority of chemolithotrophic microorganisms that are fed on the mineral salts of the rocks and basal soil and occupy ice/till veins. the englacial ecosystem. It has been reported that microorganisms enclosed in ice present very low metabolic rates, using energy only to repair damaged biomolecules, and not to grow and reproduce19. More research is nec- essary in order to defne their importance20. Mount Pond Glacier is located on an island of volcanic origin, Deception Island, South Shetland, Antarctica (Fig. 2). Volcanic eruptions have caused the black color of glacial ice, which contains remains of lava and ash. Tese elements constitute an important supply of materials that are included in the ice, and form the small niches where microorganisms are housed. Te products of the volcano in Mount Pond consist of pyroclastic rocks, lava fows and scoria21. Te main volcanic materials are basalt (rich in Mg and Ca, and in alkali oxides of Na and K) and olivine (magnesium iron silicate). All these chemical elements are abundant in the englacial ecosystem (Table 1) and can be used by microorganisms. Microbial activity may play a signifcant role in the chemical exchange between basaltic rocks and glacial meltwater. Te signifcant amounts of reduced iron in these minerals provide potential energy sources for chemolithotrophic bacteria. Furthermore, the organic matter contained in ice, that can be available for microorganisms, comes from organic remains dragged by the glacier, and from other dead microorganisms transported vertically22. Several techniques have been used to identify the microorganisms that inhabit the Antarctic glaciers. Traditionally, microscopy techniques23 were used, subsequent attempts were made to culture24 and isolate the microorganisms25, while 16S and 18S rRNA sequencing techniques have more recently been used26. So far, the temperature limits at which microorganisms in ice present an active metabolism had been studied by several methods, including the incorporation of radiolabeled compounds, measurement of ATP and ADP concentrations, measurement of nitrate and ammonium, etc.9–27. Metabolic networks in other microbial commu- nities have been elucidated by metagenomics28 and metatranscriptomic approaches. Previously, few studies have focused on the biogeochemical cycling in the englacial ecosystem. Although it had been traditionally reported that most of the microorganisms found by sequencing were not cultivable29,30, some researches have demonstrated that culturing samples in several culture media24 and their subsequent functional genomic and metaproteomic analysis31 are possible. Tis can allow both the identifcation of proteins involved in microorganism metabolism, and the identifcation of microorganisms that take part in biogeochemical cycles inside the glacier24. Functional culturomic and metaproteomic analysis are ideally suited to englacial ecosystem studies for sev- eral reasons. Firstly, species that have not yet been recognized by 16S rRNA sequencing can be identifed by the sequences of their proteins, and moreover, it is a way of verifying that microorganisms are alive and have the capacity for metabolism and growth. Taking into account that proteins are synthesized only when they are required, the detection of whole essential proteins is an indicator to detect living cells. Secondly, culturing can also increase the abundance and activity of microorganisms, enabling metaproteomic studies to model the structure of the potential microbial community. In addition, the knowledge of the proteins used by these microorganisms makes it possible to fnd out their metabolic pathways and how microorganisms are able to survive in this extreme environment. SCIENTIFIC REPORTS | (2019) 9:12158 | https://doi.org/10.1038/s41598-019-47994-9 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 2. Geological setting of Mount Pond Glacier, Antarctica. (A) Map of Antarctica showing South Shetland Islands (Credits: NASA, http://visibleearth.nasa.gov). (B) Map of Deception Island referencing Mount Pond glacier and sampling point (red arrow). (C) West front of Mount Pond Glacier and sampling point (red circle). (D) Schematic representation of the sampling site. + − − Salinity NH4 NO2 NO3 Fe Mg S Ca Cl Mn K Na Sample pH (ppt) (mM) (mM) (mM) TDNa SRPb (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) MP1 4.50 0.18 2.10 3.30 4.86 187.99 0.42 3.57 104.40 6733.10 79.20 113051.45 1.81 93.13 671.46 MP2 5.51 0.27 2.57 2.67 5.18 190.24 0.39 3.30 110.39 7845.21 80.23 125478.32 2.31 102.36 874.21 MP3 5.58 0.31 1.98 2.90 4.40 191.35 0.55 2.90 120.69 5698.19 85.26 136578.11 7.33 77.54 547.33 Average 5.20 0.25 2.22 2.96 4.81 189.86 0.45 3.26 111.83 6758.83 81.56 125035.96 3.82 91.01 697.67 (SD) 0.49 0.05 0.25 0.26 0.32 1.71 0.09 0.28 6.73 876.71 2.65 9609.81 2.49 10.24 134.73 Table 1. Chemical analysis of ice meltwater from Mount Pond Glacier. aTDN: Total dissolved nitrogen. bSRP: Soluble reactive phosphorus. In this work, we have provided